Project Summary Dry eye syndrome (DES) affects 7- 33% of the population worldwide and is one of the leading causes for individuals to seek eye care. Patients experience chronic ocular discomfort and have an increased risk for corneal ulceration. Despite its common occurrence, the pathophysiology that initiates inflammation still perplexes many clinicians and investigators making DES difficult to treat and manage. Previous studies suggest that a reduction in the tear film volume stimulates hyperomolar stress and the production of proinflammatory cytokines and matrix metalloproteinases (MMPs), therefore disrupting the ocular surface. The mechanism that initiates this inflammation is unknown and is a prime target for novel therapeutic intervention. One mechanism suggested by data from my laboratory and now others, is that toll-like receptors (TLRs) are involved in DES inflammation by stimulating the production of proinflammatory cytokines and MMPs in response to damage-associated molecular patterns (DAMPs) from stress induced molecules or pathogen- associated molecular patterns (PAMPs) from intact or degraded microbes. On the other hand, since TLRs stimulate an innate immune response against invading pathogens, they may also play a protective role by reducing microbial colonization via the production of antimicrobial peptides (AMPs) when the ocular surface is compromised from desiccation. This scenario is consistent with the clinical presentation of DES, as concomitant microbial infections are infrequent. For these reasons, investigations into the role of TLRs in DES are imperative and may lead to the development of novel therapeutic options for the treatment of DES and microbial infections. The central hypothesis of this proposal is that DAMPs are increased on the ocular surface in dry eye and can activate TLRs to increase the production of damaging proinflammatory cytokines and MMPs while also enhancing pathogen protection through the production of AMPs. This hypothesis will be tested through the following specific aims. Specific aim 1 will examine the involvement of DAMPs in DES and DES-associated conditions. Specific aim 2 will investigate the impact of TLRs on the secretion of proinflammatory cytokines and MMPs in mice with experimental dry eye (EDE) and in human ocular surface cells. Specific aim 3 will determine the involvement of TLRs in modulating the risk for microbial infection in mice with EDE. With the prevalence of DES expected to double over the next few decades and lack of definitive treatment regimes, there is a critical need to better understand the pathophysiology of DES. At the completion of these studies, the role of TLRs in ocular surface inflammation and infection in DES will be identified, providing new insight into the pathobiology of DES inflammation and infection which will aid in the development of therapeutic regimes that reduce inflammation while not increasing the risk for infection.